Non-carrier immobilization of yeast cells by genipin crosslinking for the synthesis of prebiotic galactooligosaccharides from plant-derived galactose.

Galactooligosaccharides (GOS), as mimics of human milk oligosaccharides, are important prebiotics for modulating the ecological balance of intestinal microbiota. A novel carrier-free cell immobilization method was established using genipin to cross-link Kluyveromyces lactis CGMCC 2.1494, which produced ß-galactosidase, an enzyme essential for GOS synthesis. The resulting immobilized cells were characterized as stable by thermogravimetric analysis and confirmed to be crosslinked through scanning electron microscopy analysis (SEM) and Fourier transform infrared spectroscopy (FTIR). The Km and Vmax values of ß-galactosidase in immobilized cells towards o-nitrophenyl ß-D-galactoside were determined to be 3.446 mM and 2210 µmol min-1 g-1, respectively. The enzyme in the immobilized showed higher thermal and organic solvent tolerance compared to that in free cells. The immobilized cells were subsequently employed for GOS synthesis using plant-derived galactose as the substrate. The synthetic reaction conditions were optimized through both single-factor experiments and response surface methodology, resulting in a high yield of 49.1 %. Moreover, the immobilized cells showed good reusability and could be reused for at least 20 batches of GOS synthesis, with the enzyme activity remaining above 70 % at 35 °C.

Wolbachia infection status and molecular diversity in the species of tribe Tagiadini Mabille, 1878 (Lepidoptera: Hesperiidae) collected in China.

Wolbachia, one of the most ubiquitous heritable symbionts in lepidopteran insects, can cause mitochondrial introgression in related host species. We recently found mito-nuclear discordance in the Lepidopteran tribe Tagiadini Mabille 1878 from which Wolbachia has not been reported. In this study, we found that 13 of the 46 species of Tagiadini species tested were positive for Wolbachia. Overall, 14% (15/110) of Tagiadini specimens were infected with Wolbachia and nine new STs were found from 15 isolates. A co-phylogenetic comparison, divergence time estimation and Wolbachia recombination analysis revealed that mito-nuclear discordance in Tagiadini species is not mediated by Wolbachia, but Wolbachia acquisition in Tagiadini appears to have occurred mainly through horizontal transmission rather than codivergence.

Biotransformation of Tyrosol into a Novel Valuable α-Galactoside with Increased Solubility and Improved Anti-inflammatory Activities.

Herein, tyrosol [2-(4-hydroxyphenyl) ethanol], which is rich in olive oil and red wine, was converted to a novel bioactive galactoside by enzymic glycosylation. The gene of α-galactosidase from Geobacillus stearothermophilus 23 was cloned and expressed in Escherichia coli as catalytically active inclusion bodies. The catalytically active inclusion bodies efficiently catalyzed the galactosylation of tyrosol using either melibiose or raffinose family oligosaccharides as glycosyl donors, resulting in a glycoside with 42.2 or 14.2% yields. The glycoside product was purified and identified as p-hydroxyphenethyl α-d-galactopyranoside by mass spectrometry and NMR analyses. The inclusion bodies can be recycled and reused for at least 10 batch reactions of galactoside synthesis. Moreover, the galactoside showed 11-fold increased water solubility and reduced cytotoxicity as compared to tyrosol. Also, it exhibited higher antioxidative and anti-inflammatory activities than tyrosol based on lipopolysaccharide-induced activated BV2 cells. These results provided important insights into the application of tyrosol derivatives in functional foods.

Galactooligosaccharides: Synthesis, metabolism, bioactivities and food applications.

Prebiotics are non-digestible ingredients that exert significant health-promoting effects on hosts. Galactooligosaccharides (GOS) have remarkable prebiotic effects and structural similarity to human milk oligosaccharides. They generally comprise two to eight sugar units, including galactose and glucose, which are synthesized from substrate lactose by microbial ß-galactosidase. Enzyme sources from probiotics have received particular interest because of their safety and potential to synthesize specific structures that are particularly metabolized by intestinal probiotics. Owing to advancements in modern analytical techniques, many GOS structures have been identified, which vary in degree of polymerization, glycosidic linkage, and branch location. After intake, GOS adjust gut microbiota which produce short chain fatty acids, and exhibit excellent biological activities. They selectively stimulate the proliferation of probiotics, inhibit the growth and adhesion of pathogenic bacteria, alleviate gastrointestinal, neurological, metabolic and allergic diseases, modulate metabolites production, and adjust ion storage and absorption. Additionally, GOS are safe and stable, with high solubility and clean taste, and thus are widely used as food additives. GOS can improve the appearance, flavor, taste, texture, viscosity, rheological properties, shelf life, and health benefits of food products. This review systemically covers GOS synthesis, structure identifications, metabolism mechanisms, prebiotic bioactivities and wide applications, focusing on recent advances.

Recent innovations in immobilization of ß-galactosidases for industrial and therapeutic applications.

Immobilized enzymes are better suited for industrial applications than free enzymes due to their favorable properties such as ease of separation and reuse, and enhanced stability and storage life. ß-Galactosidases are an important class of glycosidases with hydrolysis and transglycosylation activities, which are applied in industries for lactose hydrolysis and prebiotics synthesis worldwide. The recent innovations in immobilized ß-galactosidases have improved the performance of the immobilized enzymes and broadened their applications in the fields of food, energy, and medicine. Innovations in ß-galactosidase immobilization methods include rational adsorption based on enzyme features, layer by layer adsorption for strengthened ionic bonding, 3-D printing for rapid and elaborate entrapment, modifications of either materials or enzymes for ingenious covalent binding, nontoxic crosslinking, carrier-free immobilization, and oriented immobilization either through protein engineering or enzyme display on cells, membranes, and phages, along with innovations in carrier materials involving the introduction of graphene derivatives, polyaniline nanomaterials, nanofibers, nucleotide molecules, Langmuir-Blodgett films, and so on. These innovations have partially solved the problems associated with traditional methods, resulting in enzymes with highly retained activity, excellent stability, reduced microbial contamination, enzyme leakage, and reagent toxicity. The immobilized ß-galactosidases with potential economic and environmental benefits have been extendedly used for hydrolysis of prodrugs for disease treatment, assembly of biosensors for lactose detection, synthesis of bioactive carbohydrates, and even production of food additives and industrial products, such as tagatose and bioethanol. This review describes the innovations in ß-galactosidases immobilization and the applications of these immobilized enzymes. It not only enables the fully understanding of ß-galactosidases, but also provides a valuable reference for the immobilization of other industrially-important enzymes.

Carrier-Free Immobilization of α-Galactosidase as Nano-Biocatalysts for Synthesizing Prebiotic α-Galacto-Oligosaccharides.

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%.